Fluidic systems and methods for analyses

ABSTRACT

Fluidic systems and methods for analyses are provided. In some embodiments, systems and methods for improved measurement of absorbance/transmission through fluidic systems are described. Specifically, in one set of embodiments, optical elements are fabricated on one side of a transparent fluidic device opposite a series of fluidic channels. The optical elements may guide incident light passing through the device such that most of the light is dispersed away from specific areas of the device, such as intervening portions between the fluidic channels. By decreasing the amount of light incident upon these intervening portions, the amount of noise in the detection signal can be decreased when using certain optical detection systems.

RELATED APPLICATIONS

This is a continuation application of U.S. patent application Ser. No.14/316,069, filed Jun. 26, 2014, and entitled “Structures forControlling Light Interaction with Microfluidic Devices,” which is acontinuation application of U.S. patent application Ser. No. 13/898,028,filed May 20, 2013, and entitled “Structures for Controlling LightInteraction with Microfluidic Devices,” which is a divisionalapplication of U.S. patent application Ser. No. 13/490,055, filed Jun.6, 2012, and issued as U.S. Pat. No. 8,480,975 on Jul. 9, 2013, andentitled “Structures for Controlling Light Interaction with MicrofluidicDevices,” which is a continuation of U.S. patent application Ser. No.12/698,451, filed Feb. 2, 2010 and issued as U.S. Pat. No. 8,221,700 onJul. 17, 2012, and entitled “Structures for Controlling LightInteraction with Microfluidic Devices,” which claims priority under 35U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/149,253,filed Feb. 2, 2009, and entitled “Structures for Controlling LightInteraction with Microfluidic Devices,” each of which is incorporatedherein by reference in its entirety for all purposes.

FIELD OF INVENTION

The present invention relates generally to microfluidic systems, andmore specifically, to systems and methods for controlling lightinteraction with microfluidic devices.

BACKGROUND

Optical analysis of fluids plays an important role in fields such aschemistry, microbiology and biochemistry. These fluids may includeliquids or gases and may provide reagents, solvents, reactants, orrinses to chemical or biological processes. While various microfluidicmethods and devices, such as microfluidic assays, can provideinexpensive, sensitive and accurate analytical platforms, carrying outaccurate optical measurements (e.g., absorbance or transmission) on amicrofluidic system can be challenging. Optical measurements ofmicrochannels may require, for example, time-consuming alignmentprocedures. In addition, optical noise produced by light incident uponareas outside the channels may degrade the quality of the detectedsignal through the channels. Accordingly, advances in the field thatcould reduce costs, simplify use, and/or improve optical detection inmicrofluidic systems would be beneficial.

SUMMARY OF THE INVENTION

Systems and methods for controlling light interaction with microfluidicdevices are provided. The subject matter of the present inventioninvolves, in some cases, interrelated products, alternative solutions toa particular problem, and/or a plurality of different uses of one ormore systems and/or articles.

In one set of embodiments, a series of fluidic devices are provided. Inone particular embodiment, a fluidic device comprises an articleincluding first and second opposing sides and first and secondmicrofluidic channel segments, each integral to the first side of thearticle. The fluidic device also includes an intervening portionpositioned substantially between the first and second microfluidicchannel segments, and a first optical element integral to the secondside of the article and positioned substantially between the first andsecond channel segments, and opposite the intervening portion. The firstoptical element is adapted and arranged such that when a portion of thearticle is exposed to light at a first intensity, the first opticalelement redirects at least a portion of the light away from theintervening portion, such that the intervening portion is not exposed tothe light or is exposed to the light at a second intensity lower than anintensity of the light at the intervening portion absent the firstoptical element.

In another embodiment, a fluidic device comprises an article comprisingfirst and second sides, a first microfluidic channel segment integral tothe first side of the article, and first and second optical elements,each integral to the second side of the article, wherein the firstmicrofluidic channel segment is positioned substantially between thefirst and second optical elements. A cover is positioned over the firstmicrofluidic channel segment so as to substantially enclose the firstmicrofluidic channel segment. Furthermore, an intervening surfaceportion at the second side of the article is positioned substantiallybetween the first and second optical elements, the intervening surfaceportion being substantially parallel to a surface portion of the coverthat substantially encloses the first microfluidic channel segment.

In another embodiment, a fluidic device comprises an article comprisingfirst and second sides, and first and second microfluidic channelsegments, each integral to the first side of the article. The fluidicdevice also includes a first substantially triangular optical elementintegral to the second side of the article and positioned substantiallybetween the first and second channel segments.

In some instances, the first and/or second microfluidic channel segmentsdescribed above and herein are sections of a microfluidic channelcomprising a meandering configuration including multiple turns, eachturn of the meandering channel being a different channel segment.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIGS. 1A-1E include schematic diagrams of a device including opticalelements that can be used to control light interaction on or within thedevice, according to one set of embodiments;

FIGS. 2A-2B include schematic cross-sectional diagrams of devicesshowing light interactions in the devices, according to one set ofembodiments;

FIGS. 3A-3C include, according to one set of embodiments, schematicdiagrams of channel configurations in certain devices;

FIGS. 4A-4B include cross-sectional diagrams illustrating a fabricationprocess, according to one set of embodiments;

FIG. 5 includes a plot of optical density as a function of dyeconcentration, according to one set of embodiments;

FIGS. 6A-6D include cross-sectional schematic diagrams and associatedplots of transmitted light as a function of detector position, accordingto one set of embodiments;

FIGS. 7A-7C include cross-sectional schematic diagrams, opticalmicrographs, and a plot of optical density as a function of dyeconcentration, according to one set of embodiments;

FIGS. 8A-8C include schematic diagrams illustrating various sensorlayouts, according to one set of embodiments;

FIGS. 9A-9D include plots of transmitted light as a function of sensorposition, according to one set of embodiments; and

FIG. 10 includes a plot of optical density as a function of dyeconcentration, according to one set of embodiments.

DETAILED DESCRIPTION

Systems and methods for improved measurement of absorbance/transmissionthrough fluidic systems are described. Specifically, in one set ofembodiments, optical elements are fabricated on one side of atransparent fluidic device opposite a series of fluidic channels. Theoptical elements may guide incident light passing through the devicesuch that most of the light is dispersed away from specific areas of thedevice, such as intervening portions between the fluidic channels. Bydecreasing the amount of light incident upon these intervening portions,the amount of noise in the detection signal can be decreased when usingcertain optical detection systems. In some embodiments, the opticalelements comprise triangular grooves formed on or in a surface of thedevice. The draft angle of the triangular grooves may be chosen suchthat incident light normal to the surface of the device is redirected atan angle dependent upon the indices of refraction of the external medium(e.g., air) and the device material.

Advantageously, certain optical elements described herein may befabricated along with the fluidic channels of the device in one step,thereby reducing the costs of fabrication. Furthermore, in some casesthe optical elements do not require alignment with a detector and,therefore, facilitate assembly and/or use by an end user. Otheradvantages are described in more detail below.

Additional techniques may be employed to reduce the amount of straylight transmitted through the fluidic device. For example, in someinstances, the widths of the intervening portions between the channelsegments may be reduced. Also, the light source may be arranged suchthat light is emitted only over portions of the device that lie abovethe channel segments. Both of these techniques may reduce the amount oflight transmitted through the intervening portions, thus improving thequality of the optical image. In some embodiments, the fluidic devicemay include a detector array arranged such that the areas of the arrayunder the channel segments are sensitive to light, while the other areasof the array are not.

The systems and methods described herein may find application in avariety of fields. In some cases, the systems and methods can be used toimprove the optical performance of any microfluidic system such as, forexample, microfluidic point-of-care diagnostic platforms, microfluidiclaboratory chemical analysis systems, optical monitoring systems in cellcultures or bio-reactors, among others. Optical measurements inmicrofluidic systems may be used to monitor any suitable chemical and/orbiological reaction as it takes place, diagnostic or otherwise. As aspecific example, an optical measurement step can be used during DNAsynthesis to verify the yield of each base addition (e.g., opticaltrityl monitoring) and during some forms of PCR amplification to monitorthe process.

Previous systems, such as those described in International PatentPublication No. WO2006/113727 (International Patent Application SerialNo. PCT/US2006/014583), filed Apr. 19, 2006 and entitled “FluidicStructures Including Meandering and Wide Channels,” have made use of ameandering microchannel to image a two-dimensional space. For example, amicrofluidic channel may be in the form of a tight “S” shape havingmultiple channel segments, forming an area of about 2 mm, e.g., a“measurement area” including both channel and non-channel regions. Incertain embodiments, this measurement area does not require finealignment for optical measurements (unlike a single straight channel)and forms a measurement area which can be easily interrogated optically.For instance, a detector may be positioned over all or a portion of themeasurement area made up of channel and non-channel regions. Onelimitation of the use of meandering structures in the context oftransmission measurement, though, is that some of the light shiningthrough these measurement areas will pass through the interveningportions between the microfluidic channel segments (that is, thenon-channel regions). This light may reach the optical detector withoutreflecting changes in the optical density of the contents of themicrochannel. This “stray light” can reduce the overall performance ofthe optical detection. This effect may be particularly problematic whenmaking measurements of channels with high levels of optical density. Alarge amount of stray light on the detector may wash out any changes insmall amounts of light passing through the microchannels.

The inventors have discovered within the context of the invention thatthe amount of light that passes through an intervening portion betweenmicrofluidic channels or channel segments may be reduced orsubstantially eliminated by fabricating, in the device, at least oneoptical element. The optical element may redirect at least a portion ofthe light away from the intervening portion, such that the interveningportion is not exposed to the light or is exposed to the light at asecond intensity lower than an intensity of light to which theintervening portion would be exposed in the absence of the opticalelement. The incorporation of optical elements into microfluidic channelsystems enhances the performance of the detection system, allowing theuse of simplified optics without compromising the quality of the opticalmeasurements.

Furthermore, the systems and methods described herein may be used toimprove alignment in micro-scale optical detection systems. Certainmethods for optical detection/measurements in microsystems arechallenging in that they require accurate alignment of the optics withmicro-scale features (e.g., microchannels). Such alignment can beperformed manually (e.g., with a microscope and micrometric stage) in alabor-intensive fashion, or in an automated manner (e.g., using complexrobotic positioning systems). These techniques, however, often require askilled and attentive operator or expensive, delicate automation, makingthem suboptimal for certain applications. The ability of the opticalelements to redirect light away from one or more intervening portionsbetween microfluidic channel segments may eliminate or reduce the needfor such complicated alignment procedures.

Additionally, the positioning of a detector over a measurement areawithout the need for precision is an advantage, since external (andpossibly, expensive) equipment such as microscopes, lenses, andalignment stages may not be required. Instead, alignment can beperformed by eye, or by low-cost methods that may not require analignment step by the user. For example, in one embodiment, a fluidicdevice comprising one or more optical elements and a measurement areaincluding both channel and non-channel regions can be placed in a simpleholder (i.e., in a cavity having the same shape as the fluidic device),and the measurement area can be automatically aligned with a beam oflight of the detector.

It should be noted that the systems and methods described herein may beused for guiding light in any suitable system utilizing microfabricatedstructures, and are not limited to microfluidic systems and/or thespecific channel configurations described herein.

Additional advantages of devices including optical elements constructedto redirect light are described in more detail below.

The articles, systems, and methods described herein may be combined withthose described in International Patent Publication No. WO2005/066613(International Patent Application Serial No. PCT/US2004/043585), filedDec. 20, 2004 and entitled “Assay Device and Method”; InternationalPatent Publication No. WO2005/072858 (International Patent ApplicationSerial No. PCT/US2005/003514), filed Jan. 26, 2005 and entitled “FluidDelivery System and Method”; International Patent Publication No.WO2006/113727 (International Patent Application Serial No.PCT/US06/14583), filed Apr. 19, 2006 and entitled “Fluidic StructuresIncluding Meandering and Wide Channels”; U.S. patent application Ser.No. 12/113,503, filed May 1, 2008 and entitled “Fluidic Connectors andMicrofluidic Systems”; U.S. patent application Ser. No. 12/196,392,filed Aug. 22, 2008, entitled “Liquid containment for integratedassays”; U.S. patent application Ser. No. 12/428,372, filed Apr. 22,2009, entitled “Flow Control in Microfluidic Systems”; U.S. Patent Apl.Ser. No. 61/263,981, filed Nov. 24, 2009, entitled “Fluid Mixing andDelivery in Microfluidic Systems”; and U.S. patent application Ser. No.12/640,420 filed on Dec. 17, 2009 and entitled, “Improved ReagentStorage in Microfluidic Systems and Related Articles and Methods,” eachof which is incorporated herein by reference in its entirety for allpurposes. In addition, U.S. Provisional Patent Application Ser. No.61/149,253, filed Feb. 2, 2009, entitled “Structures for ControllingLight Interaction with Microfluidic Devices,” is incorporated herein byreference in its entirety for all purposes.

Examples of fluidic devices and methods associated therewith are nowprovided.

FIGS. 1A-1E show various portions of a fluidic device including opticalelements that can be used to control light interaction on or within thedevice. FIG. 1A shows a cross section and FIG. 1B shows a perspectiveview of a fluidic device 10 which includes an article 12 having a firstsurface 14 and a second surface 16, as well as a first side 20 and asecond side 22.

As used herein, “first and second sides” of an article generally refersto the relative orientation of two portions of the article. First andsecond sides may refer to first and second surfaces of the article, orto a portion of the article that does not encompass a surface, e.g., aportion of the article that is embedded within the bulk of the article.For example, first and second microfluidic channel segments that aresaid to be integral to the first side of the article may be integral toa surface at the first side of the article or embedded within thearticle at the first side. FIG. 1A also shows the first side opposingthe second side. Two sides are said to be “opposing” when they aresubstantially parallel to each other and separated by a distance.

As shown illustratively in FIGS. 1A and 1C, first side 20 includes aplurality of channel segments (first 26, second 28, and third 30) formedtherein. A channel segment refers to a portion of a fluidic channel thatspans an entire cross-section of the channel and has a lengthsubstantially parallel to fluid flow. A channel segment may have anysuitable length, e.g., at least 1 mm, at least 5 mm, at least 1 cm, orat least 5 cm in certain embodiments. While three channel segments areshown in FIG. 1A, systems and methods described herein may comprise anysuitable number of channel segments and may be configured in anysuitable arrangement. For instance, channel segments of a device may bea part of the same fluidic channel, or may be part of separate fluidicchannels that are not in fluid communication with one another.

In some embodiments, channel segments refer to a series of repetitiveunits of one or more channels; for example, each channel of an array ofchannels may be a channel segment. In another example, a channelincludes a plurality of reaction areas positioned in series, and eachchannel portion associated with a distinct reaction area is a channelsegment. In certain cases, channel segments are sections of a fluidicchannel having a meandering configuration, each “turn” of the meanderingchannel being a different channel segment. As used herein, a “meanderingchannel” (i.e., a channel having a meandering region) includes at leasta first segment that has a flow path in a first direction and a secondsegment that has a flow path in a second direction substantiallyopposite (e.g., greater than 135 degrees from) the first direction.Often, a meandering channel will include more than two alternatingchannel segments that extend in opposite directions. Examples ofmeandering channel regions are provided below.

In some embodiments, the two or more channel segments of a device arespaced apart from each other by intervening portions, i.e., non-channelportions. For instance, first side 20 includes intervening portions 27and 29. An intervening portion may include portions of a surface of anarticle (e.g., surface portion 14′ of FIG. 1A) and/or a portion of thearticle that does not encompass a surface (e.g., portions 15 of FIG.1A). In some embodiments, an intervening portion has one or moredimensions (e.g., a width, height, and/or length) of at least 0.5 mm, atleast 1 mm, at least 5 mm, at least 1 cm, or at least 5 cm in certainembodiments. A dimension of an intervention portion may, for example,define the distance between two channel segments.

The fluidic device illustrated in FIGS. 1A and 1C also includes a cover31 positioned over the plurality of channel segments. The cover may bepositioned over the channel segments so as to substantially enclose thechannel segments. In some instances, the cover may comprise a tape(e.g., flexible tape), glass (e.g., a cover slide), rigid plastic, orany other suitable material as described in more detail below.

In the set of embodiments illustrated in FIGS. 1A and 1C, second side 22includes a plurality of optical elements (first 32 and second 34) formedtherein. As used herein, the term “optical element” is used to refer toany feature formed or positioned on or in an article or device thatchanges the direction (e.g., via refraction or reflection), focus,polarization, and/or other property of incident electromagneticradiation relative to the light incident upon the article or device inthe absence of the element. For example, an optical element may comprisea lens (e.g., concave or convex), mirror, grating, groove, or otherfeature formed or positioned in or on an article. An article itselfabsent a unique feature, however, would not constitute an opticalelement, even though one or more properties of incident light may changeupon interaction with the article.

FIGS. 1A and 1C also show an intervening surface portion 33 between thefirst and second optical elements. As shown in FIG. 1A, the interveningsurface portion may be substantially parallel to the surface portion ofthe cover that substantially encloses the microfluidic channel segments.While two optical elements are shown in FIGS. 1A and 1C, the articlesdescribed herein may comprise any suitable number of optical elementsand any suitable number of intervening surface portions between theoptical elements. Furthermore, some articles do not include interveningsurface portions between the optical elements, e.g., a series of opticalelements may be configured to form alternating ridges and grooves in acorrugated fashion.

The optical elements described herein may be, in some cases,substantially transparent (e.g., to visible light, infrared radiation,etc.). In other embodiments, optical elements may comprise asubstantially opaque material. In some cases, optical elements maycomprise one or more reflective surfaces. For example, an opticalelement may comprise a channel, the walls of which are coated with areflective material such as a metal (e.g., Ni, Ag, Au, Pt) or asemi-conductor (e.g., Si, glass).

An optical element may comprise a groove, which may be open orsubstantially enclosed. As shown in the embodiment illustrated in FIGS.1A and 1C, optical elements 32 and 34 are in the form of grooves thatare substantially triangular; however, other shapes are also possible.For instance, in other embodiments, the cross-section of an opticalelement may be of any suitable shape such as a hemisphere, square,rectangle, trapezoid, etc. Some optical elements have a semi-sphericalor semi-ovular shape. The shape and/or angle of the groove may beselected such that incident light normal to the surface of the device isredirected away from the area directly below the groove. This mayenhance the probability that little or no light is incident uponunwanted areas around the channels (e.g., intervening portions 27 and29), reducing the amount of noise in the detection signal. Accordingly,an optical element may have any suitable size, configuration, and/orshape to achieve improvements in signal to noise, as described in moredetail below.

In some cases, an optical element may be a feature that protrudes from asurface of an article. For example, FIG. 1D includes triangular opticalelements 32 and 34 formed in the shape of a prism. It should beunderstood that “triangular” optical elements include any elements thatare triangular in cross-section, whether formed in a substrate (e.g., asin FIG. 1A) or on a substrate (e.g., as in FIG. 1D). Other shapes thatmay be formed include, for example, half-cylinders, rectangular prisms,etc.

An optical element may comprise, in some cases, one or more fluids(e.g., a dye). For example, in one set of embodiments, the opticalelement is formed as a channel (e.g., by placing a cover over surface 16of the article) and the channel is filled with a light-absorbing fluidsuch as an opaque dye. Dyes of any suitable concentration may be used.In some embodiments, the concentration of the dye may be at least about0.1 grams, at least about 0.5 grams, at least about 1 gram, at leastabout 5 grams, at least about 10 grams, at least about 50 grams, or atleast about 100 grams of dye material per mL of solvent (e.g., water).

FIG. 1C shows a schematic diagram of an optical device during operation,according to one set of embodiments. During operation, fluidic device 10is positioned between a light source 36 and an optical detector 38 suchthat first side 20 (comprising one or more channel segments) faces thedetector and second side 22 (comprising one or more optical elements)faces light source 36 and is exposed to light 42. The detector may beassociated with one or more fluidic channel segments in the fluidicdevice, e.g., to determine light transmission through one or more of thechannel segments. In one set of embodiments, when a portion of thearticle is exposed to light at a first intensity, the optical elementsredirect at least a portion of the light away from the interveningportions. For example, one or more optical elements may be adapted andarranged so as to redirect at least a portion of the light away from asurface portion of the first side, the surface portion being adjacent toat least one channel segment. In FIG. 1C, optical element 32 is adaptedand arranged to redirect light away from intervening portion 27, whichincludes surface portion 14′. Similarly, optical element 34 is adaptedand arranged to redirect light away from intervening portion 29, whichincludes surface portion 49. Redirecting light may include, forinstance, reflecting the light (e.g., away from the article), refractingthe light (e.g., through the article in a direction away from theintervening portion), or both.

One or more optical elements may be constructed and arranged to redirectat least about 10%, at least about 25%, at least about 50%, at leastabout 75%, or at least about 90% of incident light away from anintervening portion. As light is redirected, the intervening portionsare not exposed to the light or are exposed to the light at a secondintensity lower than an intensity of the light at the interveningportion absent the optical elements. For example, in some cases, atleast one optical element is adapted and arranged such that theintervening portions are exposed to the light at a second intensity atleast about 50% lower, at least about 75% lower, or at least about 90%lower than an intensity of the light at the intervening portion absentthe optical elements.

In some embodiments, one or more optical elements are adapted andarranged so as to redirect at least a portion of the light away from thecenter plane (e.g., 32′ and 34′ in FIG. 1A) of the optical element, suchthat the underlying portion of the device directly below the opticalelement is not exposed to light or is exposed to light at a secondintensity lower (e.g., about 25% lower) than the intensity to which theunderlying portion would be exposed were the optical element absent. Asused herein, a region is “directly below” an object when it lies on theside of an object opposite that which is exposed to light from thesource. The region directly below an object may span the width of theobject and the depth of the article perpendicular to the outermostsurface on either the first or second side of the object. For example,in FIG. 1C, region 52 lies directly below optical element 34.

An example of the use of optical elements to redirect light is shown inFIG. 1C. As illustrated in FIG. 1C, portion 44 of article 12 is exposedto light 42 from the light source. In some cases, the light source anddevice are oriented such that the angle of incidence of the light onsurface 16 is between about 85° and about 95°, or at substantially 90°.At surface 16, light that is incident on areas absent the opticalelements is transmitted into the bulk of the device without asubstantial change in direction, as indicated by arrows 42. Lightincident upon optical element 32, however, interacts at an anglesubstantially different than 90°. Optical element 32 redirects at leasta portion of the light away from intervening portion 27, e.g., byreflection, refraction, and/or both. Arrows 46′ represent light that isreflected away from the optical element, while arrows 46 illustratelight that is refracted through the article, but away from interveningportions between the fluidic channels. Thus, intervening portion 27 isnot exposed to the light, or is exposed to the light at an intensityless than it would have been in the absence of optical element 32.Furthermore, although not shown in this figure, light may be absorbed bythe article or redirected at other angles, thereby reducing the amountof light detected by the detector at intervening portion 27. Opticalelement 34 functions in a similar manner, in this case redirecting lightaway from intervening portion 29 and generally away from region 52directly below the optical element.

In some cases, one or more optical elements are adapted and arranged toredirect at least a portion of the incident light into one or morefluidic channels on the opposite side of the article. For example, asshown in FIG. 1C, a portion of the light incident upon optical element32 is refracted through the article and redirected into fluidic channels26 and 28. Advantageously, this can increase the amount of light used tointerrogate a sample in fluidic channels 26 and 28.

It should be understood that while much of the description and figuresherein describe the positioning of optical elements at a side of anarticle opposite the channels, in some cases the optical elements maypositioned at the same side as the channels. For instance, opticalelements 32 and 34 of FIG. 1A-1D may be formed in surface 14 and mayoptionally include a reflective surface so as to redirect light 42′ awayfrom portion 43 of the detector. In other cases, an article may includea combination of optical elements formed in or on both surfaces of thearticle. The geometry of the device and configuration of features may bechosen, in some cases, such that any light passing through the bulk ofthe article from a first side is redirected toward the channels on theopposite side of the article. The design of a system with opticalelements may be undertaken with the goal of both reflecting light andre-directing light away from the intervening portions between channels.Without wishing to be bound by theory, the design of a fluidic devicemay take into account the following:

The trajectory of refracted light is determined by Snell's Law:

n ₁ sin(β₁)=n ₂ sin(β₂)  [1]

where n₁ and n₂ are the indices of refraction of the medium in which thelight originates and is transmitted, respectively, β₁ is the anglebetween the angle of incidence and the normal at the interface, and β₂is the angle of refraction, as outlined in FIG. 1E.

Design features that may be varied to increase the amount of lightredirected away from the intervening portions include, for example, thewidth of the channel (W), the pitch of the optical elements (P1), thepitch of the channels (P2), the depth of the channel (D), the width ofthe optical elements (V), the draft angle of the optical elements (α),the thickness of the microfluidic substrate (T), the index of refractionof microfluidic substrate (n₂), the index of refraction of externalmedium (n₁), and the incident angle of light on the substrate (assumeperpendicular to substrate).

For example, FIG. 2A includes device 210 comprising substrate 211 inwhich channels 212, 213, and 214 and optical elements 216, 217, 218 and219 are formed. The thickness of the substrate is illustrated bydimension 220 in FIG. 2A. The width of a channel is measured as thewidest cross-sectional dimension of the channel substantially parallelto the surface in which it is formed. For example, the width of channel213 is indicated by dimension 221 in FIG. 2A. The half-width of channel214 is indicated by dimension 222. Similarly, the width of an opticalelement is also measured as the widest cross-sectional dimension of theelement substantially parallel to the surface in or on which it isformed. For example, the width of optical element 218 is indicated bydimension 224 in FIG. 2A. The depths of the channels, as indicated bydimension 226 in FIG. 2A, are measured perpendicular to the surface inwhich they are formed.

The pitch of two channels is measured as the distance between a firstpoint on a first channel and a second point on a second channel, whereinthe first and second points are located in similar positions withintheir respective channels. In other words, the pitch is equal to thewidth of a channel plus the gap between that channel and the adjacentchannel. For example, in FIG. 2A, the pitch of channels 213 and 214 maybe measured as the distance between similar edges of the channels, asindicated by dimension 230. In some embodiments, the pitches of alladjacent channels are substantially constant, as indicated in FIG. 2A;however, in other cases the pitches between channels may vary. The pitchof two optical elements is measured in a similar manner as shown, forexample, by dimension 232 in FIG. 2A. In some embodiments, the pitchesof all adjacent optical elements may be substantially constant, or mayvary, e.g., depending the particular light interaction desired.

To minimize stray light, improved results are obtained in someembodiments when the pitch (P1) of the optical elements matches thepitch of the channels (P2). The width of the optical elements (V) may bechosen such that the area between the optical elements (P-V) is lessthan the width of the channel (W). As (P-V) decreases relative to W, thepercentage of incident light that is redirected by the optical elementsincreases. To increase the amount of light redirected away from theintervening portions by the optical elements, the thickness of thesystem may be set so that light refracted by the optical elements isdirected onto the channels. Since there may be multiple channels, theremay be multiple preferred thicknesses for the system.

One may create a model to calculate preferred thicknesses by imaginingan incident light ray (e.g., perpendicular to the article) striking thearticle halfway between the bottom and the edge of the optical element(see, for example, light ray 240 in FIG. 2A). The thickness of thearticle may be selected such that this light reaches the center of achannel. To determine this thickness, one may begin by calculating theangle of the refracted light to the vertical within the substrate (β).This angle is a function of the angle of incidence, the draft angle ofthe optical element, and the angle of refraction. Examining thegeometry, one can see:

β₁=90 deg.−α  [2]

θ=90 deg.−α−β₂=β₁−β₂  [3]

Using Snell's Law (Equation 1), the angle of refraction (β₂) can becalculated as:

$\begin{matrix}{{\beta_{2} = {\sin^{- 1}\left( {\frac{n_{1}}{n_{2}}\sin \; \beta_{1}} \right)}}{{Therefore}\text{:}}} & \lbrack 4\rbrack \\{\theta = {{90{\deg.{- \alpha}}} - {\sin^{- 1}\left( {\frac{n_{1}}{n_{2}}{\sin \left( {90{\deg.{- \alpha}}} \right)}} \right)}}} & \lbrack 5\rbrack\end{matrix}$

If one were to assume a draft angle (α) of 35.3°, an article refractiveindex (n₂) of 1.57 (e.g., polystyrene), and a refractive index of air(n₁) of roughly 1.0, the internal angle of refraction would be 23.4°.

Following this ray of from the center of the side of the optical elementto the center of a channel, this angle can be used to calculate anintermediate measure of thickness (t):

$\begin{matrix}{{\tan \; \theta} = \frac{x}{t}} & \lbrack 6\rbrack\end{matrix}$

The distance (x) from the point below the incident light and the centerof a the closest channel is half the pitch minus the distance betweenthe bottom of the optical element and the edge (V/4). The center of anyadditional channel is a multiple of the pitch. Note that in FIG. 2A, thethickness was chosen to direct the refracted light onto a channel twochannels away (n=2) from the closest channel below the incident light.In FIG. 2B, n=3.

$\begin{matrix}{x = {\left( {\frac{P}{2} - \frac{V}{4}} \right) + {nP}}} & \lbrack 7\rbrack\end{matrix}$

This yields:

$\begin{matrix}{t = \frac{\left( {\frac{P}{2} - \frac{V}{4}} \right) + {nP}}{\tan \; \theta}} & \lbrack 8\rbrack\end{matrix}$

The total thickness of the substrate also includes the depth of thechannels and half the depth of the triangular optical elements. Thus, apreferred thickness for a device including triangular optical elementsand multiple channel segments can be calculated as:

$\begin{matrix}{T = {t + D + \frac{\left( {V/2} \right)}{\tan \; \alpha}}} & \lbrack 9\rbrack\end{matrix}$

Example 2 includes a description of experiments performed using a devicedesigned in this manner.

FIG. 2B includes a ray trace image generated by Mathematica (Lenslabplug-in) of a proposed design of a system including optical elementspositioned above microfluidic channels. In FIG. 2B, device 310 comprisesarticle 312 in which channels 314 and optical elements 316 are formed.Light rays 318 are directed toward surface 320 of the article, where aportion of the light is refracted through the article. The device isconstructed and arranged such that the light is directed away fromintervening portions 322, and toward channels 314 and ultimatelydetector components 324. Note that in some cases, light that is incidentupon a channel does not necessarily interact with the detector at aposition directly below the channel. For example, light rays 330interact with channel 314′ and detector component 324′, which is notdirectly below (indicated by region 332) channel 314′.

It should be understood that while triangular optical elements are shownin FIGS. 1-2, a similar analysis can be performed with devices havingoptical elements of other shapes and configurations.

Light scattering or stray light may be reduced by fabricating the wallsof these optical elements to be very smooth. In some embodiments, theroot mean square (RMS) surface roughness may be, for example, less thanabout 1 μm. In other embodiments, the RMS surface roughness may be lessthan about 0.8 μm, less than about 0.5 μm, less than about 0.3 μm, orless than about 0.1 μm. RMS surface roughness is a term known to thoseskilled in the art, and may be expressed as:

$\sigma_{h} = {\left\lbrack {\langle\left( {z - z_{m}} \right)^{2}\rangle} \right\rbrack^{1/2} = \left\lbrack {\frac{1}{A}{\int_{A}{\left( {z - z_{m}} \right)^{1/2}{A}}}} \right\rbrack^{1/2}}$

where A is the surface to be examined, and |z−z_(m)| is the local heightdeviation from the mean. Substantial roughness on the surface of anoptical element may result in unwanted scattering or redirection oflight at an undesired angle.

As described herein, optical elements may have various shapes, sizes andconfigurations. For example, in one set of embodiments, the largestcross-sectional dimension of an optical element is at least about 300microns, 500 microns, 700 microns, 1 mm, 1.5 mm, 2 mm, or greater(typically, less than 1 cm). In some embodiments, the largestcross-sectional dimension of an optical element is its width. Forinstance, as shown in FIG. 2A, the largest cross-sectional dimension ofoptical element 218 is its width 224.

In some cases, e.g., as illustrated in FIG. 2A, at least one opticalelement (e.g., optical element 218) is positioned between first andsecond channel segments (e.g., segments 213 and 214, respectively), andthe optical element has a largest cross-sectional dimension (e.g., width224) greater than or equal to the width of an intervening portionpositioned substantially between the first and second channel segments,but less than the combination of the widths of the two channel segmentsand the width of the intervening portion.

Optical elements may, in some cases, span at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, or at least about 90%of the length of one or more channel segments on or in the article. Forexample, in FIG. 1B, optical elements 32 and 34 span the entire lengthof channel segments 26, 28, and 30.

In some fluidic devices described herein, one or more optical elementsand/or channels have a non-zero draft angle. As known to those ofordinary skill in the art, a draft angle is the amount of taper, e.g.,for molded or cast parts, perpendicular to the parting line. Forexample, as shown in FIG. 3A, a substantially rectangular channel 110,which has walls 112-A and 112-B that are substantially perpendicular tosurface 114 (e.g., a parting line), has a draft angle 116 of 0°. Thecross sections of fluidic channels having non-zero draft angles, on theother hand, may resemble a triangle, a parallelogram, a trapezoid, etc.For example, as shown in the embodiment illustrated in FIG. 3B, channel120 has a substantially triangular cross-section. Draft angle 116 isformed by the angle between a line perpendicular to surface 114 and wall127-A of the channel, and is non-zero in this embodiment.

The draft angle of an optical element, channel, or other component maybe, for example, between about 1° and about 40°, between about 1° andabout 30°, between about 1° and about 20°, between about 1° and about10°, between about 2° and about 15°, between about 3° and about 10°, orbetween about 3° and about 8°. For instance, the draft angle may begreater than or equal to about 1°, 2°, 3°, 4°, 5°, 6°, 7°, 8°, 9°, or10°, 20°, 37.5°, or 40°. In some cases, it is desirable for opticalelements or channels to have specific draft angles so that they arecompatible with a certain detection technique.

As described herein, optical elements may be combined with a fluidicdevice comprising one or more meandering channels. As shown in a topview of the illustrative embodiment of FIG. 3C, channel 208 includes ameandering (e.g., serpentine) region having a tightly packed channelsystem having a series of turns 210 and channel segments 212 that spanover a large area (A) relative to the width of the channel. The areaspanned by the meandering channel (i.e., the area of the meanderingregion) is the area bound by outermost points of the meandering channelalong each axis, shown approximately in FIG. 3C by the dashed lines.This area may constitute a measurement area over which a detector may bepositioned, the measurement area including both channel segments 212 andintervening channel portions 220 (i.e., non-channel segments).

FIG. 3C also shows plurality of optical elements 236 interspersedbetween the channel segments at the intervening channel portions 220.One measurement area may include, for example, greater than or equal to3, 5, 8, 10, 15, 20, 30, 40, or 50 optical elements. The opticalelements may be the same or different from one another, and may have anysuitable shape or size as described herein. Furthermore, as illustrated,a fluidic device may include optical elements that extend past themeasurement area and/or past the channel segments. This configurationmay allow control of light even at turns 210 of the channel segments.

As shown in FIG. 3C, the length of channel segments 212-A and 212-C arethe same. In other embodiments, however, the lengths of the segments ofthe meandering channel vary within the channel. Channel segments havingdifferent lengths may result in a measurement area having differentshapes. The meandering channel (and the area of the channel) can bedesigned to have any suitable shape, e.g., a square, rectangular,circular, oval, triangular, spiral, or an irregular shape, since incertain cases the overall shape does not affect the fluid flowconditions within the channel.

In FIG. 3C, the area (A) that the meandering channel spans is defined bythe surface area given by dimension B times (x) dimension C. Typically,the area spanned by the channel (i.e., as viewed from above the channel,perpendicular to the direction of fluid flow) is on the order ofmillimeters squared (mm²). For instance, the area may be greater than orequal to 0.5 mm², greater than or equal to 1 mm², greater than or equalto 2 mm², greater than or equal to 5 mm², greater than or equal to 10mm², or greater than or equal to 50 mm². However, in other embodiments,e.g., depending on the method used for detection, the area spanned by ameandering channel may be between 0.25 mm² and 0.5 mm², or between 0.1mm² and 0.25 mm². Typically, the area spanned by the meandering channelis designed to be relatively large (e.g., on the order of mm²) comparedto conventional microfluidic systems, so that a wide area can be usedfor detection and so the total amount of signal that can be detected isincreased, especially in combination with one or more optical elementsdescribed herein.

In some embodiments, the optical elements described herein are integralto a surface of the article. As used herein, “integral” refers to acondition of being a single, unitary construction, as opposed toseparate parts that are connected by other means. For instance, integraloptical elements of article may be formed in a surface of the article.Integral optical elements may be either concave or convex relative tothe surface on which they are formed. For example, optical elements 32and 34 in FIGS. 1A-1B are shown as concave optical elements integral tosurface 16. In some cases, such optical elements, or molds for theoptical elements, are fabricated using an photolithography process,e.g., as shown in FIGS. 4A-4B and as described in more detail below.

As shown in various embodiments herein, one or more optical elements maybe positioned substantially between two channel segments and/or one ormore channel segments may be positioned substantially between twooptical elements. A first object is said to be positioned “substantiallybetween” second and third other objects when substantially all of thefirst object lies between the center planes of the second and thirdobjects. As used herein, a “center plane” of an object refers to animaginary plane that intersects the geometric center of the crosssection of the object and is substantially perpendicular to thesubstrate in or on which the object is positioned or formed. The term“geometric center” (or “centroid”) is given its normal meaning in theart. For example, in FIG. 1A, channels 26, 28, and 30 comprise centerplanes 26′, 28′, and 30′, which intersect geometric centers 26″, 28″ and30″, respectively. In addition, optical elements 32 and 34 comprisecenter planes 32′ and 34′ which intersect geometric centers 32″ and 34″,respectively. Optical element 32 is positioned substantially betweenchannels 26 and 28, and optical element 34 is positioned substantiallybetween channels 28 and 30. Channel segment 28 is positionedsubstantially between optical elements 32 and 34.

In some embodiments, one or more optical elements of a device lie on asubstantially different plane than one or more channels of the device.For example, in FIG. 1A, the plane 60 intersecting the central axes ofthe optical elements (positioned at the geometric centers and extendingout of and into the page) does not intersect the plane 62 intersectingthe central axes of the channels. In some embodiments, no line drawnbetween any first point on or within a first microfluidic channelsegment and any second point on or within a second microfluidic channelsegment intersects any point on or within an optical element. In someinstances, no line drawn between any first point on or within a firstoptical element and any second point on or within a second opticalelement intersects any point on or within a microfluidic channelsegment.

In other embodiments, however, all or a portion of an optical elementlies on the same plane as one or more channel or channel segment. Forinstance, an optical element may be formed in or on the same surface asthe channels. In another example, an optical element is formed on a sideopposite a channel, but extends such that a plane perpendicular to thesurface of the article passes through both the channel and the opticalelement. In some cases, a line drawn between a first point on or withina first channel segment and a second point on or within a second channelsegment intersects a point on or within the optical element.

Fluidic devices described herein comprising optical elements may beoptionally combined with other features (e.g., certain detectionsystems, lenses, etc.) for reducing the amount of stray light and/or forincreasing the signal to noise ratio. FIGS. 5-10 show various examplesof detection systems and results of experiments performed when suchsystems were used in combination with devices described herein. In somecases, however, these features may be implemented independently of theoptical elements described herein.

In some embodiments, additional techniques may be employed thatcompensate for the transmission of stray light through the microfluidicdevice. For example, the size (e.g., width, surface area, volume) of theintervening portions in the system may be reduced, thus reducing thepercentage of light incident on the intervening portions. It should benoted that while it may not be practical to eliminate the interveningportions between the channels, as discussed in International PatentPublication No. WO2006/113727, thinner intervening portions and/or widerfluidic channels may result in less stray light transmitted and,therefore, improved performance.

The effects of reducing the size of the intervening portions on theamount of transmitted stray light can be evaluated by measuringtransmission or absorbance in the system when the microchannels arefilled with a perfectly absorbent fluid. Transmission through such asystem is calculated as:

$\begin{matrix}{{Trans} = \frac{I}{I_{o}}} & \lbrack 10\rbrack\end{matrix}$

where I₀ is the intensity of light transmitted with a perfectly clear(index matched) fluid in the channels, and I is the intensity of lighttransmitted with a perfectly absorbent fluid in the channel.

The optical density (OD) is a measure of absorbance in such a system,which is calculated as the negative log of transmission:

OD=−log(Trans)  [11]

A system with a minimum amount of stray light transmissions results in alarge OD. In theory, a measurement zone filled with a perfectlyabsorbent fluid and with no intervening portions and no stray lightwould have a transmission of 0% and very large OD. In practice, it isdifficult to completely eliminate stray light in any system. Atransmission measurement through an extremely absorbent fluid in amicrowell (no walls, or even channels) might be 0.01%, yielding an OD of4. In general, though, transmission measurements below 1% can bedifficult to achieve. A reasonable range of ODs that may be achieved maybe within the range of about 0 to about 2.

Assuming a perfectly absorbent fluid in the channels, the transmissionthrough a meandering channel region (without optical elements to blockor re-direct light) is simply a function of the width of the interveningportions and the width of the channel. For example, in a system withintervening portions with widths of x and channels with widths of y, theminimum transmission would be x/(x+y). In the case of a meanderingchannel with identical widths for all intervening portions and channels,the value of x/(x+y) is 50% (yielding a maximum OD of 0.3). Similarly, asystem with channels twice the width of the intervening portions wouldyield a minimum transmission of 33% (a maximum OD of 0.477). There is anupper (and lower) range for the channel widths based on the flowrequired in the system, since an increase in width of the channelresults in an increase in cross section and changes in the properties ofthe channels, such as a reduction in the resistance to flow. Likewise,there is a lower range at which intervening portions can be reliablyfabricated (e.g., depending on the fabrication technique). Example 3outlines a set of experiments in which the widths of the interveningportions were varied.

In some embodiments, a detection system includes measuring the lighttransmitted through the channel portions independently of the lighttransmitted through the intervening portions. For example, one may imagethe measurement area with a digital camera, measure the intensity oflight on the pixels that correspond to the channels and discard thepixels corresponding to the channel walls or the intervening portions.Optionally, lenses may be incorporated to focus the image on the planeof the channels. Such a measurement system could potentially deliverextremely high performance (avoiding stray light) and yield maximum ODsgreater than about 2, e.g., OD=2-4.

However, in some cases, including a camera/imaging system may result ina relatively high cost of the imaging device, relatively high cost oflenses, precision required in positioning and alignment, robustness toshock or environmental conditions, and implementation of software toidentify which pixels are to be measured and which to be ignored.Accordingly, these factors may be weighed with their benefits and may besuitable for certain, but not all, applications.

In one embodiment, a relatively inexpensive and robust imaging systemwas developed for a channel system utilizing a linear image sensor. Alinear image sensor is a one-dimensional array of multiple small opticaldetectors which can be individually measured. FIGS. 8A and 8B includeschematic diagrams of exemplary imaging systems used to measuretransmission through meandering channels. The optical detector 810 inFIG. 8A is a single photodiode that may image a substantial portion ofthe meandering channel. In FIG. 8B, on the other hand, the opticaldetector 812 comprises a linear image sensor that measures only portionsof the meandering channel. Optionally, optical components such as acollimating lens for the light source and/or a focusing lens to transmitthe image to the linear image sensor (not shown) may be utilized toimprove imaging. FIG. 8C includes a micrograph of a meandering channelused in one set of embodiments. A typical measurement area for a linearimage sensor is indicated as region 820. In certain devices, eachoptical detector of a linear image sensor can be measured individually.Such a system can be used to measure transmission through only a portionof a system. For example, a linear image sensor located under ameandering channel region as shown in FIG. 8C can be used to measurelight transmission only through the channels. To do this, only thereadings from detectors under the channels are recorded. Other detectorspositioned under non-channel portions (e.g., intervening portions) andstruck by stray light can be ignored. In this manner, linear imagesensors can be used to selectively measure light transmission throughchannels in a meandering channel region, eliminating the problem ofstray light, and yielding accurate transmission/absorbance measurementsfor the microfluidic system.

Examples of linear image sensors include the Hamamatsu 59227, a 6.4 mmlong array of 512, 250-micron wide pixels at 12.5 micron spacing, theFairchild Imaging CMOS 1421, a 14.5 mm-long array of 2048, 7-micron widepixels with 7 um center-to-center spacing, and the Panavision SVILIS-500, a 3.9 mm long array of 500, 62.5-wide pixels with 7.8 umcenter-to-center spacing. Example 4 outlines the use of a linear imagesensor in conjunction with the devices and methods described herein.

In some cases, the system may be designed to eliminate potential straylight before it reaches the fluidic device. For example, stray light maybe eliminated by creating a light source that includes a geometry thatmatches the pattern of channel(s), directing light only onto thechannels and away from the channel walls or intervening portions.

A variety of determination (e.g., measuring, quantifying, detecting, andqualifying) techniques may be used with devices described herein.Determination techniques may include optically-based techniques such aslight transmission, light absorbance, light scattering, light reflectionas well as luminescence techniques such as photoluminescence (e.g.,fluorescence), chemiluminescence, bioluminescence, and/orelectrochemiluminescence. Those of ordinary skill in the art know how tomodify microfluidic devices in accordance with the determinationtechnique used. For instance, for devices including chemiluminescentspecies used for determination, an opaque and/or dark background may bepreferred. For determination using metal colloids, a transparentbackground may be preferred. Furthermore, any suitable detector may beused with devices described herein. For example, simplified opticaldetectors, as well as conventional spectrophotometers and opticalreaders (e.g., 96-well plate readers) can be used.

When more than one chemical and/or biological reaction (e.g., amultiplex assay) is performed on a device, the signal acquisition can becarried out by moving a detector over each analysis region. In analternative approach, a single detector can detect signal(s) in each ofthe analysis regions simultaneously. In another embodiment, an analyzercan include, for example, a number of parallel opticalsensors/detectors, each aligned with a analysis region and connected tothe electronics of a reader. Additional examples of detectors anddetection methods are described in more detail in U.S. patentapplication Ser. No. 12/196,392, filed Aug. 22, 2008, entitled “Liquidcontainment for integrated assays”, which is incorporated herein byreference.

As described herein, a meandering channel of an analysis region may beconfigured and arranged to align with a detector such that uponalignment, the detector can measure a single signal through more thanone adjacent channel segments of the meandering channel. In someembodiments, the detector is able to detect a signal within at least aportion of the area of the meandering channel and through more than onesegments of the meandering channel such that a first portion of thesignal, measured from a first segment of the meandering channel, issimilar to a second portion of the signal, measured from a secondsegment of the meandering channel. In such embodiments, because thesignal is present as a part of more than one segment of the meanderingchannel, there is no need for precise alignment between a detector andan analysis region.

Additional examples and descriptions of detection systems are providedin the Examples section.

In some embodiments, the fluidic devices described herein include areaction site in fluid communication with one or more channels orchannel segments. For example, the fluidic device may comprise areaction site comprising a binding partner (e.g., an antibody, antigen,etc.) associated with a surface of a channel segment. An entity in thefluid flowing in the channel segment may interact (e.g., bind,chemically react, etc.) with the binding partner, and the interactionmay be optically detectable.

In one set of embodiments, a fluidic device described herein is used forperforming an immunoassay. The immunoassay may be, for example, a directimmunoassay, a sandwich (e.g., 2-site) immunoassay, or a competitiveimmunoassay, as known to those of ordinary skill in the art. Certaindevices may include a combination of one or more such immunoassays.

In one particular embodiment, a fluidic device is used for performing animmunoassay (e.g., for human IgG or PSA) and, optionally, uses sliverenhancement for signal amplification. A device described herein may haveone or more similar characteristics as those described in U.S. patentapplication Ser. No. 12/113,503, filed May 1, 2008 and entitled “FluidicConnectors and Microfluidic Systems”, which is incorporated herein byreference. In such an immunoassay, after delivery of a sample containinghuman IgG to a reaction area or analysis region, binding between thehuman IgG and anti-human IgG can take place. One or more reagents, whichmay be optionally stored in the device prior to use, can then flow overthis binding pair complex. One of the stored reagents may include asolution of metal colloid (e.g., a gold conjugated antibody) thatspecifically binds to the antigen to be detected (e.g., human IgG). Thismetal colloid can provide a catalytic surface for the deposition of anopaque material, such as a layer of metal (e.g., silver), on a surfaceof the analysis region. The layer of metal can be formed by using a twocomponent system: a metal precursor (e.g., a solution of silver salts)and a reducing agent (e.g., hydroquinone), which can optionally bestored in different channels prior to use.

As a positive or negative pressure differential is applied to thesystem, the silver salt and hydroquinone solutions can merge at achannel intersection, where they mix (e.g., due to diffusion) in achannel, and then flow over the analysis region. Therefore, ifantibody-antigen binding occurs in the analysis region, the flowing ofthe metal precursor solution through the region can result in theformation of an opaque layer, such as a silver layer, due to thepresence of the catalytic metal colloid associated with theantibody-antigen complex. The opaque layer may include a substance thatinterferes with the transmittance of light at one or more wavelengths.Any opaque layer that is formed in the microfluidic channel can bedetected optically, for example, by measuring a reduction in lighttransmittance through a portion of the analysis region (e.g., ameandering channel region) compared to a portion of an area that doesnot include the antibody or antigen. Alternatively, a signal can beobtained by measuring the variation of light transmittance as a functionof time, as the film is being formed in a analysis region. The opaquelayer may provide an increase in assay sensitivity when compared totechniques that do not form an opaque layer. Additionally, variousamplification chemistries that produce optical signals (e.g.,absorbance, fluorescence, glow or flash chemiluminescence,electrochemiluminescence), electrical signals (e.g., resistance orconductivity of metal structures created by an electroless process) ormagnetic signals (e.g., magnetic beads) can be used to allow detectionof a signal by a detector.

It should be understood that devices described herein may be used forany suitable chemical and/or biological reaction, and may include, forexample, other solid-phase assays that involve affinity reaction betweenproteins or other biomolecules (e.g., DNA, RNA, carbohydrates), ornon-naturally occurring molecules. In some embodiments, a chemicaland/or biological reaction involves binding. Different types of bindingmay take place in devices described herein. The term “binding” refers tothe interaction between a corresponding pair of molecules that exhibitmutual affinity or binding capacity, typically specific or non-specificbinding or interaction, including biochemical, physiological, and/orpharmaceutical interactions. Biological binding defines a type ofinteraction that occurs between pairs of molecules including proteins,nucleic acids, glycoproteins, carbohydrates, hormones and the like.Specific examples include antibody/antigen, antibody/hapten,enzyme/substrate, enzyme/inhibitor, enzyme/cofactor, bindingprotein/substrate, carrier protein/substrate, lectin/carbohydrate,receptor/hormone, receptor/effector, complementary strands of nucleicacid, protein/nucleic acid repressor/inducer, ligand/cell surfacereceptor, virus/ligand, etc. Binding may also occur between proteins orother components and cells. In addition, devices described herein may beused for other fluid analyses (which may or may not involve bindingand/or reactions) such as detection of components, concentration, etc.

Non-limiting examples of analytes that can be determined using fluidicdevices described herein include specific proteins, viruses, hormones,drugs, nucleic acids and polysaccharides; specifically antibodies, e.g.,IgD, IgG, IgM or IgA immunoglobulins to HTLV-I, HIV, Hepatitis A, B andnon A/non B, Rubella, Measles, Human Parvovirus B19, Mumps, Malaria,Chicken Pox or Leukemia; human and animal hormones, e.g., thyroidstimulating hormone (TSH), thyroxine (T4), luteinizing hormone (LH),follicle-stimulating hormones (FSH), testosterone, progesterone, humanchorionic gonadotropin, estradiol; other proteins or peptides, e.g.troponin I, c-reactive protein, myoglobin, brain natriuretic protein,prostate specific antigen (PSA), free-PSA, complexed-PSA, pro-PSA,EPCA-2, PCADM-1, ABCA5, hK2, beta-MSP (PSP94), AZGP1, Annexin A3, PSCA,PSMA, JM27, PAP; drugs, e.g., paracetamol or theophylline; markernucleic acids, e.g., PCA3, TMPRS-ERG; polysaccharides such as cellsurface antigens for HLA tissue typing and bacterial cell wall material.Chemicals that may be detected include explosives such as TNT, nerveagents, and environmentally hazardous compounds such as polychlorinatedbiphenyls (PCBs), dioxins, hydrocarbons and MTBE. Typical sample fluidsinclude physiological fluids such as human or animal whole blood, bloodserum, blood plasma, semen, tears, urine, sweat, saliva, cerebro-spinalfluid, vaginal secretions; in-vitro fluids used in research orenvironmental fluids such as aqueous liquids suspected of beingcontaminated by the analyte. In some embodiments, one or more of theabove-mentioned reagents is stored in a channel or chamber of a fluidicdevice prior to first use in order to perform a specific test or assay.

Some embodiments of the invention are in the form of a kit that mayinclude, for example, a microfluidic system, a source for promotingfluid flow (e.g., a vacuum), and/or one, several, or all the reagentsnecessary to perform an analysis except for the sample to be tested. Insome embodiments, the microfluidic system of the kit may have aconfiguration similar to one or more of those shown in the figuresand/or as described herein. The fluidic device of the kit may beportable and may have dimensions suitable for use in point-of-caresettings.

The kit may include reagents and/or fluids that may be provided in anysuitable form, for example, as liquid solutions or as dried powders. Insome embodiments, a reagent is stored in the microfluidic system priorto first use, as described in more detail herein. When the reagents areprovided as a dry powder, the reagent may be reconstituted by theaddition of a suitable solvent, which may also be provided. Inembodiments where liquid forms of the reagent are provided, the liquidform may be concentrated or ready to use. The fluids may be provided asspecific volumes (or may include instructions for forming solutionshaving a specific volume) to be flowed in the microfluidic system.

The kit may be designed to perform a particular analysis such as thedetermination of a specific disease condition. For instance, markers(e.g., PSA) for specific diseases (e.g., prostate cancer) may beincluded (e.g., stored) in a device or kit in a fluid or dry form priorto first use of the device/kit. In order to perform a particularanalysis or test using the kit, the fluidic device may be designed tohave certain geometries, and the particular compositions, volumes, andviscosities of fluids may be chosen so as to provide optimal conditionsfor performing the analysis in the system. For example, if a reaction tobe performed at an analysis region requires the flow of an amplificationreagent over the analysis region for a specific, pre-calculated amountof time in order produce an optimal signal, the fluidic device may bedesigned to include a channel segment having a particularcross-sectional area and length to be used with a fluid of specificvolume and viscosity in order to regulate fluid flow in a predeterminedand pre-calculated manner. Washing solutions and buffers may also beincluded. The device may optionally include one or more reagents storedtherein prior to first use. Furthermore, the kit may include a device orcomponent for promoting fluid flow, such as a source of vacuumdimensioned to be connected to an outlet. The device or component mayinclude one or more pre-set values so as to create a known (andoptionally constant) pressure drop between an inlet and an outlet of thefluidic device. Thus, the kit can allow one or more reagents to flow fora known, pre-calculated amount of time at an analysis region, or atother regions of the system, during use. Those of ordinary skill in theart can calculate and determine the parameters necessary to regulatefluid flow using general knowledge in the art in combination with thedescription provided herein.

A kit described herein may further include a set of instructions for useof the kit. The instructions can define a component of instructionalutility (e.g., directions, guides, warnings, labels, notes, FAQs(“frequently asked questions”), etc., and typically involve writteninstructions on or associated with the components and/or with thepackaging of the components for use of the microfluidic system.Instructions can also include instructional communications in any form(e.g., oral, electronic, digital, optical, visual, etc.), provided inany manner such that a user will clearly recognize that the instructionsare to be associated with the components of the kit.

In some embodiments, microfluidic systems described herein containstored reagents prior to first use of the device and/or prior tointroduction of a sample into the device. In some cases, one or both ofliquid and dry reagents may be stored on a single article. Additionallyor alternatively, the reagents may also be stored in separate vesselssuch that a reagent is not in fluid communication with the microfluidicsystem prior to first use. The use of stored reagents can simplify useof the microfluidic system by a user, since this minimizes the number ofsteps the user has to perform in order to operate the device. Thissimplicity can allow microfluidic systems described herein to be used byuntrained users, such as those in point-of-care settings, and inparticular, for devices designed to perform immunoassays. It has beendemonstrated previously that the storage of the reagents in the form ofliquid plugs separated by air gaps were stable for extended periods oftime (see, for example, International Patent Publication No.WO2005/072858 (International Patent Application Serial No.PCT/US2005/003514), filed Jan. 26, 2005 and entitled “Fluid DeliverySystem and Method,” which his incorporated herein by reference in itsentirety). Fluidic devices for storing reagents may also include aconfiguration as described in U.S. patent application Ser. No.12/640,420 filed on Dec. 17, 2009 and entitled, “Improved ReagentStorage in Microfluidic Systems and Related Articles and Methods,” whichis incorporated herein by reference in its entirety. In otherembodiments, however, microfluidic devices described herein do notcontain stored reagents prior to first use of the device and/or prior tointroduction of a sample into the device.

As used herein, “prior to first use” of the device means a time or timesbefore the device is first used by an intended user after commercialsale. First use may include any step(s) requiring manipulation of thedevice by a user. For example, first use may involve one or more stepssuch as puncturing a sealed inlet to introduce a reagent into thedevice, connecting two or more channels to cause fluid communicationbetween the channels, preparation of the device (e.g., loading ofreagents into the device) before analysis of a sample, loading of asample onto the device, preparation of a sample in a region of thedevice, performing a reaction with a sample, detection of a sample, etc.First use, in this context, does not include manufacture or otherpreparatory or quality control steps taken by the manufacturer of thedevice. Those of ordinary skill in the art are well aware of the meaningof first use in this context, and will be able easily to determinewhether a device of the invention has or has not experienced first use.In one set of embodiments, devices of the invention are disposable afterfirst use, and it is particularly evident when such devices are firstused, because it is typically impractical to use the devices at allafter first use.

The devices described herein may comprise one or more channels orchannel segments. A “channel” or “channel portion”, as used herein,means a feature on or in an article or substrate (e.g., formed in asurface/side of an article or substrate) that at least partially directsthe flow of a fluid. A channel, channel portion, or channel segment,etc. can have any cross-sectional shape (circular, oval, triangular,irregular, square or rectangular, trapezoidal, or the like) and can becovered or uncovered. In embodiments where it is covered, at least oneportion of the channel can have a cross-section that is substantiallyenclosed, or the entire channel may be substantially enclosed along itsentire length with the exception of its inlet(s) and outlet(s). In somecases, the inlet and/or outlet may also be enclosed or sealed, e.g., toprevent fluids and/or other reagents from being removed from the device(e.g., due to evaporation).

A channel, channel segment, channel portion, etc., may also have anaspect ratio (length to average cross-sectional dimension) of at least2:1, more typically at least 3:1, 5:1, or 10:1 or more. In someembodiments, one or more channels, channel segments, channel portions,intervening channels, etc., is microfluidic. “Microfluidic,” as usedherein, refers to a device, apparatus or system including at least onefluid channel having a cross-sectional dimension of less than 1 mm, anda ratio of length to largest cross-sectional dimension of at least 3:1.A “microfluidic channel” or “microfluidic channel segment” as usedherein, is a channel meeting these criteria. Though in some embodiments,devices of the invention may be microfluidic, in certain embodiments,the invention is not limited to microfluidic systems and may relate toother types of fluidic systems. Furthermore, it should be understoodthat all or a majority of the channels described herein may bemicrofluidic in certain embodiments. The “cross-sectional dimension”(e.g., a diameter, a height, and/or a width) of a channel, channelsegment, channel portion, or intervening channel, etc. is measuredperpendicular to the direction of fluid flow. In one set of embodiments,the maximum cross-sectional dimension of one or more channels or channelsegments containing embodiments described herein are less than about 750microns, less than about 500 microns, less than about 300 microns, lessthan about 200 microns, less than about 100 microns, less than about 50microns, less than about 25 microns, less than about 10 microns, or lessthan about 5 microns. In some cases, at least two cross-sectionaldimensions (e.g., a height and a width) of a channel, channel segment,or channel portion have one or more of the dimensions listed above(e.g., a width of less than 500 microns and a height of less than 200microns).

One or more channels or channel segments described herein may have anysuitable length. In some cases, the channels or channel segments may beat least about 1 mm long, at least about 2 mm long, at least about 5 mmlong, at least about 10 mm long, at least about 20 mm long, at leastabout 50 mm long, or longer.

The channels or channel segments may also be spaced any suitabledistance apart from each other. For example, in some cases, the width ofone or more intervening portions between channels or channel segmentsmay be less than about 5 mm, less than about 2 mm, less than about 1 mm,less than about 500 microns, less than about 300 microns, less thanabout 200 microns, less than about 100 microns, less than about 50microns, less than about 25 microns, less than about 10 microns, lessthan about 5 microns, or less. In certain embodiments, channel segmentsmay be separated by a distance of less than 0.01 times, less than 0.1times, less than 0.25 times, less than 0.5 times, less than 1 times,less than 2 times, less than 5 times, or less than 10 times the averagelargest width of the channel segment.

The channels or channel segments may also be oriented in any suitablemanner. In some instances, all channels or channel segments are spaced asubstantially equal distance from each other (i.e., the widths of theintervening portions are all substantially the same). The channels orchannel segments may also be oriented such that two or more (e.g., all)are substantially parallel to each other.

In some cases the dimensions of a channel may be chosen such that fluidis able to freely flow through the article or substrate. The dimensionsof the channel may also be chosen, for example, to allow a certainvolumetric or linear flowrate of fluid in the channel. Of course, thenumber of channels and the shape of the channels can be varied by anymethod known to those of ordinary skill in the art. In some cases, morethan one channel or capillary may be used.

In some embodiments described herein, microfluidic systems include onlya single interconnected channel with, for example, less than 5, 4, 3, 2,or 1 channel intersection(s) when in use. A layout based on a singlechannel with minimal or no intersections may be reliable because thereis only one possible flow path for any fluid to travel across themicrofluidic chip.

A microfluidic system described herein may have any suitable volume forcarrying out a chemical and/or biological reaction or other process. Theentire volume of a microfluidic system includes, for example, anyreagent storage areas, reaction areas, liquid containment regions, wasteareas, as well as any fluid connectors, and microfluidic channelsassociated therewith. In some embodiments, small amounts of reagents andsamples are used and the entire volume of the microfluidic system is,for example, less than 10 milliliters, less than 5 milliliters, lessthan 1 milliliter, less than 500 microliters, less than 250 microliters,less than 100 microliters, less than 50 microliters, less than 25microliters, less than 10 microliters, less than 5 microliters, or lessthan 1 microliter.

A fluidic device and/or an article described herein may be portable and,in some embodiments, handheld. The length and/or width of the deviceand/or article may be, for example, less than or equal to 20 cm, 15 cm,10 cm, 8 cm, 6 cm, or 5 cm. The thickness of the device and/or articlemay be, for example, less than or equal to 5 cm, 3 cm, 2 cm, 1 cm, 8 mm,5 mm, 3 mm, 2 mm, or 1 mm. Advantageously, portable devices may besuitable for use in point-of-care settings.

All or a portion of a fluidic device such as an article or a cover canbe fabricated of any suitable material. For example, articles thatinclude channels may be formed of a suitable for forming a microchannel.Non-limiting examples of materials include polymers (e.g., polyethylene,polystyrene, polymethylmethacrylate, polycarbonate,poly(dimethylsiloxane), PTFE, PET, and a cyclo-olefin copolymer), glass,quartz, and silicon. The article and/or cover may be hard or flexible.Those of ordinary skill in the art can readily select a suitablematerial based upon e.g., its rigidity, its inertness to (e.g., freedomfrom degradation by) a fluid to be passed through it, its robustness ata temperature at which a particular device is to be used, itstransparency/opacity to light (e.g., in the ultraviolet and visibleregions), and/or the method used to fabricate features in the material.For instance, for injection molded or other extruded articles, thematerial used may include a thermoplastic (e.g., polypropylene,polycarbonate, chlorotrifluoroethylene, acrylonitrile-butadiene-styrene,nylon 6), an elastomer (e.g., polyisoprene, isobutene-isoprene, nitrile,neoprene, ethylene-propylene, hypalon, silicone), a thermoset (e.g.,epoxy, unsaturated polyesters, phenolics), or combinations thereof. Insome embodiments, the material and dimensions (e.g., thickness) of anarticle and/or cover are chosen such that it is substantiallyimpermeable to water vapor. For instance, a fluidic device designed tostore one or more fluids therein prior to first use may include a covercomprising a material known to provide a high vapor barrier, such asmetal foil, certain polymers, certain ceramics and combinations thereof.In other cases, the material is chosen based at least in part on theshape and/or configuration of the device. For instance, certainmaterials can be used to form planar devices whereas other materials aremore suitable for forming devices that are curved or irregularly shaped.

In some instances, a fluidic device is formed of a combination of two ormore materials, such as the ones listed above. For instance, thechannels of the device may be formed in a first material (e.g.,poly(dimethylsiloxane)), and a cover that is formed in a second material(e.g., polystyrene) may be used to seal the channels. In anotherembodiment, a first set of channels is formed in a first articlecomprising a first material and a second set of channels is formed in asecond article comprising a second material. In yet another embodiment,channels of the device may be formed in polystyrene or other polymers(e.g., by injection molding) and a biocompatible tape may be used toseal the channels. The biocompatible tape may include a material knownto improve vapor barrier properties (e.g., metal foil, polymers or othermaterials known to have high vapor barriers). A variety of methods canbe used to seal a microfluidic channel or portions of a channel, or tojoin multiple layers of a device, including but not limited to, the useof adhesives (such as acrylic or silicone based adhesives), use adhesivetapes, gluing, bonding, lamination of materials, or by mechanicalmethods (e.g., clamping).

Sealing a channel and/or any inlets and outlets may protect and retainany gases, liquids, and/or dry reagents that may be stored within achannel. In addition or alternatively to one or more covers describedherein, in certain embodiments, a fluid having low volatility, such asan oil or glycol may be placed in the end of a tube to help preventevaporation and/or movement of other fluids contained therein.

Devices comprising optical elements and channels (e.g., microchannels)described herein may be fabricated using a variety of techniques. Forexample, the devices described herein may be formed using injectionmolding, hot embossing, or other plastic engineering techniques. Thedevices may also be manufactured using traditional machining techniques.In some cases, the devices may be fabricated by producing a mold andtransferring the features of the mold to a hardenable polymer (e.g.,PDMS). Molds may be fabricated by, for example, etching features into asilicon wafer (e.g., via an anisotropic KOH etch) and transferring thefeatures onto a hardenable material (e.g., SU-8) which may then serve asa mold. In some cases, the microfluidic devices described herein includean article that is a single, integral piece of material without joinedlayers.

In one set of embodiments, purely photolithographic techniques are usedto fabricated the channels and optical elements in a polymer. FIGS.4A-4B illustrate a fabrication process that may be used to producetriangular optical elements in photoresist. In FIG. 4A, a layer ofphotoresist 410 overlies substrate 412. Photomask 414, comprisingUV-transparent feature 416, is exposed to ultraviolet light 418. Theultraviolet light is directed at an angle 420 from the normal of thephotomask. The development of the photoresist layer results in theformation of a triangular feature 430, as shown in FIG. 4B. Thistechnique may produce features having smooth surfaces. In addition, thetechnique may be used to fabricate features with a relatively wide rangeof draft angles (e.g., from about 0° to about 20°). Such methods areknown to those of ordinary skill in the art.

The manufacturing processes used to produce devices by injection molding(or other plastic engineering techniques, such as hot embossing), oftenrequire molds having non-zero draft angles on some or all of thefeatures to be replicated in plastic. As discussed above, a draft angleis the amount of taper for molded or cast parts perpendicular to theparting line (a square channel with walls perpendicular to the floorhaving a draft angle of zero degrees). A non-zero draft angle is oftennecessary to allow demolding of the replica from the molding tool.

The fabrication of elements with non-zero draft angles is challenging.For instance, for microfluidic structures (e.g., channels) havingvarious depths, the corresponding mold must have features with multipleheights in addition to non-zero draft angles. These types of molds canbe challenging to fabricate on the microscale, as molding microchannelsin plastic with constrictions in draft angle, depth, as well as in widthis not trivial.

In fact, few techniques can yield the appropriate shapes for a moldhaving non-zero draft angles. To widen the breadth of technologies ableto produce the appropriate shapes, an indirect route to the fabricationof the mold can be chosen. For instance, the channels themselves can becreated in various materials, by various techniques to produce a master.The negative shape of the master is then obtained (e.g., byelectrodeposition), resulting in a mold for injection molding. Thetechniques capable of yielding a master with non-zero draft angles andvarious depths include: (1) milling with one or more trapezoidal-shapedbits, (2) photolithographic techniques in combination with thickphotosensitive polymers, for instance photosensitive glass orphotoresist like SU8, in combination with a back-side exposure or atop-side exposure with light with a non-normal angle. An example of theuse of non-normal top-side exposure with photosensitive glass to producefeatures with non-zero draft angles is described in U.S. Pat. No.4,444,616. The preparation of multiple depths can be achieved bymultiple photolithographic exposures onto multiple layers ofphotosensitive material. (3) KOH etching on silicon substrates can alsoproduce non-zero draft angles, according to the crystalline planes ofthe silicon. (4) Alternative to straight draft angles, channels havingrounded side-walls can also produce suitable master for molds. Suchrounded side-walls can be achieved by isotropic etching onto planarsurface (e.g., HF etching on Pyrex wafers), or by reflowing structuresphotoresist by heat treatment. (5) Deep Reactive Ion Etching (DRIE) canalso produce non-zero degree draft angles under certain parameters.

The following examples are intended to illustrate certain embodiments ofthe present invention, but are not to be construed as limiting and donot exemplify the full scope of the invention.

Example 1 Fabrication of Microfluidic Channels

A method for fabricating a microfluidic channel system is described.

Channel systems, such as the ones shown in FIGS. 1A and 1B, weredesigned with a computer-aided design (CAD) program. The microfluidicdevices were formed in poly(dimethylsiloxane) Sylgard 184 (PDMS, DowCorning, Ellsworth, Germantown, Wis.) by rapid prototyping using mastersmade in SU8 photoresist (MicroChem, Newton, Mass.). The masters wereproduced on a silicon wafer and were used to replicate the negativepattern in PDMS. The masters contained two levels of SU8, one level witha thickness (height) of ˜70 μm defining the channels in the immunoassayarea, and a second thickness (height) of ˜360 μm defining the reagentstorage and waste areas. Another master was designed with channel havinga thickness (height) of 33 μm. The masters were silanized with(tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane (ABC-R, Germany).PDMS was mixed according to the manufacturer's instructions and pouredonto the masters. After polymerization (4 hours, 65° C.), the PDMSreplica was peeled off the masters and access ports were punched out ofthe PDMS using stainless steel tubing with sharpened edges (1.5 mm indiameter). To complete the fluidic network, a flat substrate such as aglass slide, silicon wafer, polystyrene surface, flat slab of PDMS, oran adhesive tape was used as a cover and placed against the PDMSsurface. The cover was held in place either by van der Waals forces, orfixed to the microfluidic device using an adhesive.

In other embodiments, the microfluidic channels were made in polystyreneor other thermoplastics by injection molding. This method is known tothose of ordinary skill in the art. The volume of an injection moldingcavity can be defined by a bottom surface and a top surface separated bya hollow frame which determines the thickness of the molded article. Foran article including channel features and or other microscale elementson two opposing sides of the article, the bottom and top surfaces of themolding cavity may include raised features that create the channelfeatures on either side of the article. For an article including channelfeatures on only one side of the article, only the top or bottom surfaceof the molding cavity includes such features. Thru-holes that passthrough the entire thickness of the article can be produced by pinstraversing the cavity, embedded in one or more surfaces of the cavityand contacting the other side. For instance, the pins may extend fromonly the top surface, only the bottom surface, or from both the top andbottom surfaces.

Example 2 Performance of a System Comprising Triangular Optical Elements

This example describes the transmission profiles of systems employing ameandering channel, one with triangular optical elements (grooves) andanother without. An article was fabricated in polystyrene with identicalsystems of fluidic channels on one side. Some of these channels includedtriangular optical elements between the channels on the other side(shielded channels). Other channels did not include triangular opticalelements between them (normal/standard channels with no shielding). Thechannels were 160 microns in width. Intervening portions between thechannels were 60 microns in width. The article thickness was designedusing the model described above. Triangular optical elements were alsodesigned as described in the model above with an angle of 35.3°, a widthof 160 microns, and a pitch of 220 microns. Optical measurements wereperformed using a single collimated LED light source and a singlephotodiode detector.

Measurements were performed with an approximate index-matching liquid inthe channels (water) and with a concentrated absorbing dye (MethyleneBlue, 20 mg/ml in water). Using water in the “normal” channel (channelwithout optical elements) as the baseline, the following transmissionmeasurements were made:

Transmission OD Water in Normal Channel 100%  0.00 Dye in Normal Channel27% 0.56 Water Shielded Channel 26% 0.58 Dye in Shielded Channel  1%1.98Assuming a perfectly absorbing dye, the transmission through normalchannels should be 27%, since the channel walls make up 60/(60+160)=27%of the area of the measurement zone. Experimental results confirmed thisprediction. Note that the range of ODs provided by a non-shieldedchannel of these dimensions would be 0 to 0.56. In shielded channelswith dye, only 1% of the incident light was transmitted. The triangularoptical element was designed to either block light that would betransmitted through the intervening portions or directed the light intothe channels. The dye in the channels absorbed most, if not all, thelight striking the channels.

With water in the shielded channels, 26% of the light incident on themeasurement zone was transmitted. With a width 60 microns and a pitch of220 microns, the triangular optical elements blocked 73% of the topsurface of the measurement area. The remaining 27% of the area waspositioned directly above channels. Since these channels were filledwith index matching liquid, it was assumed that they transmitted all ofthe light striking them. A total transmission of 26% indicated that, inthis particular experiment, significantly more of the light incident onthe optical elements was reflected out of the system than was directedto the channels.

To understand the measurement range of the shielded channels, acomparison was made between the intensity of light transmitted throughthe shielded channels with dye and the intensity of light transmittedthrough the shielded channels with water. Using the shielded channelswith water as a baseline, the transmission with dye was 4%. Thisindicated that the range of ODs provided by the shielded system withchannels of these dimensions would be 0 to 1.40. This represents asignificant improvement over the normal configuration. FIG. 5 presents acomparison of ODs measured in shielded meandering channels and ODsmeasured in unshielded meandering channels. In this example,erioglaucine dye was used. As can be seen, the shielding delivered alarger dynamic range of ODs corresponding to superior performance.

A more detailed comparison of transmitted light can be obtained usingthe linear image sensor system described above. FIG. 6A includes aschematic diagram outlining light transmission through a microfluidicmeandering channel measurement zone without optical elements. In thisset of experiments, the channels were filled with dark dye (10 mg/mLeriogalucine dye). A collimated light source was used to shine incidentlight onto the meandering channel measurement zone. A focusing lens anda linear image sensor was used to detect light through the measurementzone. The light incident upon the channels was absorbed by the dye,while the light incident between the channels passed through thearticle. FIG. 6B is a plot of the transmitted light as a function ofposition across the measurement zone. The peaks in FIG. 6B indicate thepresence of a large amount of stray light between the channels.

FIG. 6C includes a schematic diagram outlining light transmissionthrough a microfluidic meandering channel measurement zone with opticalelements. As in the previous set of experiments, the channels werefilled with dye, which absorbed light incident upon the channels. FIG.6D, like FIG. 6B, includes a plot of transmitted light as a function ofposition across the measurement zone. However, in this instance, thepeaks corresponding to the positions between the channels have beenreduced dramatically, meaning stray light between the channel reduceddue to the presence of the optical elements. Due to the shieldingprovided by the optical elements, when they are employed, a singlephotosensor may provide nearly equivalent optical performance comparedto a more complex linear image sensor. This shows that simplifiedoptical systems can be used in combination with fluidic devicesdescribed herein.

Example 3 Reducing the Width of Intervening Portions

In this example, several samples with various widths of interveningportions were fabricated and tested. FIG. 7A includes a micrograph and aschematic illustration of a device comprising 120-micron-wide opticalelements spaced 100 microns apart. In FIG. 7B, the optical elements arespaced only 30 microns apart. The channels were filled with dye(Erioglaucine) at various concentrations, and measurements oftransmissions through the meandering channel region were taken. FIG. 7Cincludes a plot of the net OD as a function of the dye concentration forseveral devices including varied inter-element spacings. As can be seenfrom the plot, the OD increases with an increase in dye concentrationand a decrease in inter-element spacing. Table 1 summarizes thetheoretical maximum projected optical density (minimum transmission) andactual optical performance of these systems.

TABLE 1 Predicted and measured maximum optical densities for variousdevices. Channel Width of Inter- Predicted Measured width veningPortions Max OD Max OD 120 μm 50 μm 0.53 0.49 120 μm 60 μm 0.48 0.43 120μm 70 μm 0.43 0.38 120 μm 80 μm 0.40 0.35 120 μm 90 μm 0.37 0.32 120 μm100 μm  0.34 0.30

Example 4 Use of Linear Image Sensors

This example describes the use of a linear image sensor in conjunctionwith the systems and methods described herein.

A linear image sensor was positioned underneath a meandering channel asshown in FIG. 8B such that detection elements were positioned below thesurface including the channel. A focusing lens was mounted between thesensor and the meandering channel so that a focused image of the channelwas projected onto the sensor surface. Collimated light was used toilluminate the meandering channel. In an alternative experimental setup,the linear image sensor was placed immediately underneath the meanderingchannel (i.e., within less than 0.5 mm), alleviating the need for a lensto be placed between the meandering channel and the surface of theoptical detector.

Measurements of the system were performed with various fluids in thechannel including index-matching liquid, dye diluted in water, andconcentrated dye. FIGS. 9A-9D include plots of transmitted light as afunction of position along the linear image sensor for various dyeconcentrations. In FIG. 9A, a low dye concentration (0.05 mg/mLerioglaucine) was used in the channel. FIGS. 9B-9D show dyeconcentrations of 0.4 mg/mL, 1.6 mg/mL, and 50 mg/mL respectively. Lesslight was transmitted through the channels (i.e., absorbance increased)as the dye concentration increased. A software program was written toidentify which pixels corresponded to positions within the channel.Selecting only these pixels, transmission was calculated as:

$\begin{matrix}{T = \frac{\begin{matrix}{{Intensity}\mspace{14mu} {of}\mspace{14mu} {light}\mspace{14mu} {detected}} \\{{with}\mspace{14mu} {target}\mspace{14mu} {liquid}\mspace{14mu} {channel}}\end{matrix}}{\begin{matrix}{{Intensity}\mspace{14mu} {of}\mspace{14mu} {light}\mspace{14mu} {detected}\mspace{14mu} {with}} \\{{index}\mspace{14mu} {matching}\mspace{14mu} {liquid}\mspace{14mu} {in}\mspace{14mu} {channel}}\end{matrix}}} & \lbrack 12\rbrack\end{matrix}$

A total transmission value was calculated by averaging the measurementsfrom all the identified channel pixels.

Various concentrations of dyes were imaged in the channels(corresponding to various levels of absorption in the channels).Transmissions were calculated using the method explained above andconverted into ODs. FIG. 10 includes plots of OD as a function of dyeconcentration when using a single photodetector (measuring the lighttraveling throughout the channels and between the channels) and whenusing a linear image sensor (discriminating pixels). The linear imagesensor delivered a larger dynamic range of ODs corresponding to superiorperformance.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

What is claimed is:
 1. (canceled)
 2. A multiplex assay systemcomprising: an article supporting solid-phase assays, said articlecomprising a rigid planar substrate comprising two or more differentliquid containment regions, each liquid containment region comprising atleast one analysis region that can be interrogated optically, eachanalysis region having one or more binding partners associated with asurface of the substrate, wherein said binding partners bind one or moreanalytes present in a sample in the liquid containment region, andwherein, for at least one liquid containment region, the one or moreanalytes is selected from the group consisting of prostate specificantigen (PSA), free PSA, complexed-PSA, pro-PSA and human kallikreinprotein (hk2).
 3. The multiplex assay system of claim 2, wherein theprotein binding partner is an antibody.
 4. The multiplex assay system ofclaim 2, wherein the fluid sample obtained from a whole blood, bloodserum, or blood plasma sample.
 5. The multiplex assay system of claim 2further comprising an optical device configured to detect light from theanalysis region.
 6. The multiplex assay system of claim 2, wherein eachdifferent liquid containment region is a chamber having a depth measuredperpendicular to a surface of the rigid planar substrate and across-sectional dimension substantially parallel to the surface of therigid planar substrate.
 7. The multiplex assay system of claim 6,wherein the chamber is integral to a surface of the rigid planarsubstrate.
 8. The multiplex assay system of claim 6, wherein the chambercomprises a separate structure connected to the rigid planar substrate.9. The multiplex assay system of claim 8, wherein the separate structureis connected to the rigid planar substrate through an adhesive and/orvan der Waals forces.
 10. The multiplex assay system of claim 9, whereinthe adhesive is an acrylic or silicone based adhesive.
 11. The multiplexassay system of claim 6, wherein the chamber is covered.
 12. Themultiplex assay system of claim 6, wherein the chamber is uncovered. 13.The multiplex assay system of claim 2, wherein each different liquidcontainment region is configured to contain less than 1 milliliter ofliquid.
 14. The multiplex assay system of claim 5, wherein the opticaldevice comprises a light source and a detector.
 15. The multiplex assaysystem of claim 5, wherein the optical detection system furthercomprises a camera or imaging system.
 16. The multiplex assay system ofclaim 14, wherein the detector is configured to detectphotoluminescence, fluorescence, chemiluminescence, bioluminescence,and/or electrochemiluminescence.
 17. The multiplex assay system of claim2, wherein the rigid planar substrate is a polyethylene, polystyrene,polymethylmethacrylate, polycarbonate, poly(dimethylsiloxane),polytetrafluoroethylene, polyethylene terephthalate, or cyclo-olefincopolymer substrate.
 18. The multiplex assay system of claim 17, whereinthe rigid planar substrate is a poly(dimethylsiloxane) substrate. 19.The multiplex assay system of claim 2, wherein the rigid planarsubstrate is a glass, quartz, or silicon substrate.
 20. The multiplexassay system of claim 2, further comprising a second article comprisinga rigid planar substrate comprising at least one analysis region inwhich one or more nucleic acid binding partners are associated with asurface of the substrate of the second article.